Natural Variation in RNA m⁶A Methylation and Its Relationship with Translational Status

N -methyladenosine (m⁶A) is the most abundant modification of eukaryotic mRNA. Although m⁶A has been demonstrated to affect almost all aspects of RNA metabolism, its global contribution to the post-transcriptional balancing of translational efficiency remains elusive in plants. In this study, we performed a parallel analysis of the transcriptome-wide mRNA m⁶A distribution and polysome profiling in two maize (Zea mays) inbred lines to assess the global correlation of m⁶A modification with translational status. m⁶A sites are widely distributed in thousands of protein-coding genes, confined to a consensus motif and primarily enriched in the 3' untranslated regions, and highly coordinated with alternative polyadenylation usage, suggesting a role of m⁶A modification in regulating alternative polyadenylation site choice. More importantly, we identified that the m⁶A modification shows multifaceted correlations with the translational status depending on its strength and genic location. Moreover, we observed a substantial intraspecies variation in m⁶A modification, and this natural variation was shown to be partly driven by gene-specific expression and alternative splicing. Together, these findings provide an invaluable resource for ascertaining transcripts that are subject to m⁶A modification in maize and pave the way to a better understanding of natural m⁶A variation in mediating gene expression regulation.